Project Summary Immune responses in mucosal and barrier tissues are critical for protecting an individual from the myriad pathogens that infect these sites, and long-term protection can be maintained in situ by subsets of T lymphocytes called tissue-resident memory T cells (TRM). At birth, humans emerge from a largely sterile environment to one where they rapidly encounter multiple, diverse antigens, particularly in the respiratory and digestive tracts. The lungs and intestines are also sites where infants are highly susceptible to infectious pathogens and can develop life-threatening diseases. This vulnerability of infants to infectious disease has been previously attributed to the immaturity of their immune systems; however, the distinct properties of infant immune responses are not fully defined, and have been largely characterized in circulation or lymphoid tissues and not in key mucosal sites. Identifying the mechanisms by which infants can mediate anti-pathogen immune responses, generate long-term memory, and maintain homeostasis, is essential for designing new strategies for vaccination and immunomodulation at the earliest life stage. We initiated studies on infant immunity (supported by the NIAID infant program) four years ago through investigations of T cell differentiation in human infant tissue samples from organ donors, airway samples from infants with viral infection, and in a mouse model of influenza virus infection. Together our findings have revealed novel aspects of early life immunity including: 1. that infant memory T cells are found predominantly in lungs and intestines and not in the periphery, and 2. that differentiation of infant T cells lead to effector responses in tissues, but reduced generation of persisting TRM. These findings suggest an overall hypothesis that infant lungs and small intestines are major sites for early T cell priming and effector differentiation, but that such responses are limited by intrinsic differences in early nave T cell activation and differentiation. Confining effector responses to mucosal sites, may prevent hyperactivation and dissemination of effector cells to multiple tissues during the early life stages of high antigen exposure. We will address our hypotheses for the genesis of infant tissue T cell responses in two aims with coordinate investigations using novel human tissue samples and infant mouse models of infection. In Aim1, we will determine role of mucosal priming in differentiation of effector and memory T cells during infancy and test the hypothesis that in situ priming of nave T cells occurs within mucosal sites during infancy with reduced LN involvement compared to adult responses. In aim 2, we will elucidate intrinsic mechanisms for reduced development of memory T cells from infant pathogen exposure, and test the hypothesis that infant effectors are transcriptionally programmed to die rather than persist and whether modulation of transcription factor expression and/or survival factors may promote infant TRM generation. Results from the proposed studies will reveal novel mechanisms for promoting infant T cell differentiation and memory generation through tissue targeting.